The long term objective of this research is to study the biochemistry and genetics of lipoamide dehydrogenases in pseudomonads which are unique in that they produce two structurally and functionally distinct lipoamide dehydrogenases. Mammals produce a single lipoamide dehydrogenase which serves pyruvate, 2-ketoglutarate and branched chain keto acid dehydrogenase. In man, genetic deficiency of lipoamide dehydrogenase results in lactic acidosis in newborns which is manifested by rapid neurological deterioration and an early death. Escherichia coli produces a single lipoamide dehydrogenase which is the E3 subunit of pyruvate and 2-ketoglutarate dehydrogenases. In contrast, both P. putida and P. aeruginosa produce two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-glc is the only lipoamide dehydrogenase produce during growth on glucose synthetic medium and is the E3 subunit of 2-ketoglutarate dehydrogenase and the L-factor in glycine oxidation. LPD-val is produced during growth in valine synthetic medium and is the specific E3 subunit of branched chain keto acid dehydrogenase. Recent data raised the possibility that there is also a separate lipoamide dehydrogenase for pyruvate dehydrogenase in P. putida. E. coli and pig heart lipoamide dehydrogenases, human glutathione reductase and mercuric reductase are all redox-active disulfide flavoproteins and share significant regions of homology suggesting a common evolutionary pathway for these apparently disparate enzymes. Studies based on amino acid compositions suggest the LPD-glc is related to E. coli and pig heart lipoamide dehydrogenases, but not to LPD-val. LPD-val appears to be unrelated to any of the other redox-active disulfide flavoproteins, except possibly mercuric reductase. The Specific Aims of this study are to 1: Determine the primary structure of LPD-val and LPD-glc. The objective of this research will be to determine the evolutionary relationships between these two proteins and other redox-active disulfide flavoproteins. 2. Purify pyruvate dehydrogenase from P. putida to determine if there is a separate structural gene for this complex.